Processes for the convergent synthesis of calicheamicin derivatives

ABSTRACT

This invention describes processes for the convergent synthesis of calicheamicin derivatives, and similar analogs using bifunctional and trifunctional linker intermediates.

This application claims priority from U.S. Provisional Application60/775,370 filed Feb. 21, 2006 entitled “Processes For The ConvergentSynthesis of Calicheamicin Derivatives” the content of which isincorporated herein in its entirety to the extent that it is consistentwith this invention and application.

FIELD OF THE INVENTION

This invention describes processes for the convergent synthesis ofcalicheamicin derivatives, and similar analogs using bifunctional andtrifunctional linker intermediates.

BACKGROUND OF THE INVENTION

The potent family of antibacterial and antitumor agents knowncollectively as the calicheamicins or the LL-E33288 complex, aredisclosed in U.S. Pat. No. 4,970,198 (1990). The compounds contain amethyltrisulfide that can be reacted with appropriate thiols to formdisulfides while at the same time introducing a functional group such asa hydrazide or similar nucleophile. Examples of this reaction with thecalicheamicins are given in U.S. Pat. No. 5,053,394. U.S. Pat. No.5,770,701 is directed to a process for preparing targeted forms ofdisulfide compounds of the LL-E33288 complex. A linker,4-(4-acetyl-phenoxy)butanoic acid, is condensed with calicheamicinN-acetyl gamma dimethyl hydrazide to afford the carboxylicacid-hydrazone which is further treated with N-hydroxysuccinimide togive the OSu ester (N-succinimidyloxy) which is ready for conjugationwith a chosen biomacromolecule.

Previously disclosed synthetic methods for constructing calicheamicinderivatives are complicated by multiple calicheamicin containingsynthetic steps having low overall yields. The calicheamicin moiety isinherently toxic, and when already part of a synthetic targetnecessitates increased safety precautions which must be observed duringmanipulation and purification of the products of each of the syntheticsteps. The claimed process provides a method of fewer steps involvingthe calicheamicin moiety with increased yields.

SUMMARY OF THE INVENTION

Discussed herein is a process to prepare calicheamicin derivatives ofFormula (I):

wherein:Z is selected from the group consisting of

Alk¹ is a branched or unbranched alkylene chain of 2 to 6 carbon atoms;Sp¹ is selected from —S—, —O—, —CONH—, —NHCO—, and —NR′—;Z¹ is H, or alkyl of 1 to 5 carbon atoms;Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two,or three groups independently selected from alkyl of 1 to 6 carbonatoms, alkoxy of 1 to 5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms,halogen, nitro, —COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′,—O(CH₂)_(n)CONHR′, and —S(CH₂)_(n)CONHR′ or a 1,2-, 1,3-, 1,4-, 1,5-,1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionallysubstituted with one, two, three, or four groups independently selectedfrom alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 5 carbon atoms,thioalkoxy of 1 to 4 carbon atoms, halogen, nitro, —COOR′, —CONHR′,—O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′, —O(CH₂)_(n)CONHR′, and—S(CH₂)_(n)CONHR′;n is an integer from 0 to 5;R′ is a straight or branched alkyl of 1 to 5 carbon atoms optionallysubstituted by one or two groups of —OH, alkoxy of 1 to 4 carbon atoms,thioalkoxy of 1 to 4 carbon atoms;Sp is a straight or branched-chain divalent or trivalent alkyl radicalof 1 to 18 carbon atoms, divalent or trivalent aryl or heteroarylradical, divalent or trivalent cycloalkyl of 3 to 18 carbon atoms orheterocycloalkyl radical, divalent or trivalent aryl- orheteroaryl-alkyl (C₁-C₁₈) radical, divalent or trivalent cycloalkyl- orheterocyclo-alkyl-alkyl (C₁-C₁₈) radical or divalent or trivalentunsaturated alkyl radical of 2 to 18 carbon atoms, wherein heteroaryl isfuryl, thienyl, N-methylpyrrolyl, pyridinyl, N-methylimidazolyl,oxazolyl, pyrimidinyl, quinolyl, isoquinolyl, N-methylcarbazoyl,aminocoumarinyl, or phenazinyl and wherein if Sp is a trivalent radical,it can be additionally substituted by dialkylamino of 1 to 5 carbonatoms, alkoxy of 1 to 5 carbon atoms, hydroxy, or alkylthio of 1 to 5carbon atoms groups;

R₅ is —CH₃, —C₂H₅, or —CH(CH₃)₂;X is an iodine or bromine atom;R₅′ is a hydrogen or the group RCO, wherein R is hydrogen, branched orunbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy of 1 to 3 carbon atoms, or thioalkoxy of 1 to 5 carbon atoms; andQ is selected from the group consisting of —NNHCO—, —NNHCS—, —NNHCONH—,—NNHCSNH—, and —NO—;comprising the steps of:a. reacting a carboxylic acid of the formula

with a mercapto compound of the formula

H₂Q-Sp-SH

in an alcohol solvent in the presence of an alkyl carboxylic acid,alk²CO₂H where alk² is 1 to 4 carbon atoms at about 20° to 70° C. forabout 1 to 24 hours, wherein Alk¹, Sp¹, Ar, Z¹, Q, and Sp are as definedabove, to produce a bilinker-carboxylic acid of the formula, wherein themercapto compound and carboxylic acid are present in a ratio of about1.2:1

b. isolating the bilinker-carboxylic acid of step (a);c. reacting the isolated bilinker-carboxylic acid of step (b) with an atleast a 3 fold molar excess of N-hydroxysuccinimide,2,3,5,6-tetrafluorophenol, pentafluorophenol, 4-nitrophenol,2,4-dinitrophenol, or N-hydroxysulfosuccinimide in the presence of1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), orN,N′-disuccinimdyl carbonate in an inert solvent containing 0-50%N,N-dimethylformamide (DMF) to generate a trilinker-activated ester ofthe formula

where Z is hereinbefore defined;d. reacting the trilinker-activated ester formed in step with amethyltrithio antitumor antibiotic CH₃—S—S—S—W′ in the presence of abase or an organic base with a methyltrithio antitumor antibioticCH₃—S—S—S—W′ in an inert organic solvent to generate an activated esterof the formula below, wherein the trilinker-activated ester in step cand the CH₃—S—S—S—W′ are in a ratio of 3.3:1 and the temperature of thereaction is ≦5° C.

e. isolating the activated ester of step (d) and purifying to yieldantitumor antibiotics of Formula (I)

An additional process discussed includes a method of preparing antitumorantibiotics of Formula (I):

wherein:Z is selected from the group consisting of

Alk¹ is a branched or unbranched alkylene chain of 2 to 6 carbon atoms;Sp¹ is selected from —S—, —O—, —CONH—, —NHCO—, and —NR′—;Z¹ is H, or alkyl of 1 to 5 carbon atoms;Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two,or three groups independently selected from alkyl of 1 to 6 carbonatoms, alkoxy of 1 to 5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms,halogen, nitro, —COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′,—O(CH₂)_(n)CONHR′, and —S(CH₂)_(n)CONHR′ or a 1,2-, 1,3-, 1,4-, 1,5-,1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionallysubstituted with one, two, three, or four groups independently selectedfrom alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 5 carbon atoms,thioalkoxy of 1 to 4 carbon atoms, halogen, nitro, —COOR′, —CONHR′,—O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′, —O(CH₂)_(n)CONHR′, and—S(CH₂)_(n)CONHR′;n is an integer from 0 to 5;R′ is a straight or branched alkyl of 1 to 5 carbon atoms optionallysubstituted by one or two groups of —OH, alkoxy of 1 to 4 carbon atoms,thioalkoxy of 1 to 4 carbon atoms;Sp is a straight or branched-chain divalent or trivalent alkyl radicalof 1 to 18 carbon atoms, divalent or trivalent aryl or heteroarylradical, divalent or trivalent cycloalkyl of 3 to 18 carbon atoms orheterocycloalkyl radical, divalent or trivalent aryl- orheteroaryl-alkyl (C₁-C₁₈) radical, divalent or trivalent cycloalkyl- orheterocyclo-alkyl-alkyl (C₁-C₁₈) radical or divalent or trivalentunsaturated alkyl radical of 2 to 18 carbon atoms, wherein heteroaryl isfuryl, thienyl, N-methylpyrrolyl, pyridinyl, N-methylimidazolyl,oxazolyl, pyrimidinyl, quinolyl, isoquinolyl, N-methylcarbazoyl,aminocoumarinyl, or phenazinyl and wherein if Sp is a trivalent radical,it can be additionally substituted by dialkylamino of 1 to 5 carbonatoms, alkoxy of 1 to 5 carbon atoms, hydroxy, or alkylthio of 1 to 5carbon atoms groups;

R₅ is —CH₃, —C₂H₅, or —CH(CH₃)₂;X is an iodine or bromine atom;R₅′ is a hydrogen or the group RCO, wherein R is hydrogen, branched orunbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy of 1 to 3 carbon atoms, or thioalkoxy of 1 to 5 carbon atoms; andQ is selected from the group consisting of —NNHCO—, —NNHCS—, —NNHCONH—,—NNHCSNH—, and —NO—;comprising the steps of:a. reacting a carboxylic acid of the formula

with a mercapto compound of the formula

H₂Q-Sp-SH

in an alcohol solvent in the presence of an alkyl carboxylic acid,alk²CO₂H where alk² is 1 to 4 carbon atoms at about 20° to 70° C. forabout 1 to 24 hours, wherein Alk¹, Sp¹, Ar, Z¹, Q, and Sp are as definedabove, to produce a bilinker-carboxylic acid of the formula, wherein themercapto compound and carboxylic acid are present in a ratio of about1.2:1

b. isolating the bilinker-carboxylic acid of step (a);c. reacting the isolated bilinker-carboxylic acid of step (b) withCH₃—S—S—S—W′ in the presence of a base or an organic base with amethyltrithio antitumor antibiotic CH₃—S—S—S—W′ in an inert organicsolvent;d. reacting the compound of step (c) with an at least a 3 fold molarexcess of N-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol,pentafluorophenol, 4-nitrophenol, 2,4-dinitrophenol, orN-hydroxysulfosuccinimide in the presence of1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), orN,N′-disuccinimdyl carbonate in an inert solvent containing 0-50%N,N-dimethylformamide (DMF) and purifying to yield compounds of Formula(I)

An additional process includes preparation of compounds of Formula (I):

wherein:Z is selected from the group consisting of

Alk¹ is a branched or unbranched alkylene chain of 2 to 6 carbon atoms;Sp¹ is selected from —S—, —O—, —CONH—, —NHCO—, and —NR′—;Z¹ is H, or alkyl of 1 to 5 carbon atoms;Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two,or three groups independently selected from alkyl of 1 to 6 carbonatoms, alkoxy of 1 to 5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms,halogen, nitro, —COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′,—O(CH₂)_(n)CONHR′, and —S(CH₂)_(n)CONHR′ or a 1,2-, 1,3-, 1,4-, 1,5-,1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionallysubstituted with one, two, three, or four groups independently selectedfrom alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 5 carbon atoms,thioalkoxy of 1 to 4 carbon atoms, halogen, nitro, —COOR′, —CONHR′,—O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′, —O(CH₂)_(n)CONHR′, and—S(CH₂)_(n)CONHR′;n is an integer from 0 to 5;R′ is a straight or branched alkyl of 1 to 5 carbon atoms optionallysubstituted by one or two groups of —OH, alkoxy of 1 to 4 carbon atoms,thioalkoxy of 1 to 4 carbon atoms;Sp is a straight or branched-chain divalent or trivalent alkyl radicalof 1 to 18 carbon atoms, divalent or trivalent aryl or heteroarylradical, divalent or trivalent cycloalkyl of 3 to 18 carbon atoms orheterocycloalkyl radical, divalent or trivalent aryl- orheteroaryl-alkyl (C₁-C₁₈) radical, divalent or trivalent cycloalkyl- orheterocyclo-alkyl-alkyl (C₁-C₁₈) radical or divalent or trivalentunsaturated alkyl radical of 2 to 18 carbon atoms, wherein heteroaryl isfuryl, thienyl, N-methylpyrrolyl, pyridinyl, N-methylimidazolyl,oxazolyl, pyrimidinyl, quinolyl, isoquinolyl, N-methylcarbazoyl,aminocoumarinyl, or phenazinyl and wherein if Sp is a trivalent radical,it can be additionally substituted by dialkylamino of 1 to 5 carbonatoms, alkoxy of 1 to 5 carbon atoms, hydroxy, or alkylthio of 1 to 5carbon atoms groups;

R₅ is —CH₃, —C₂H₅, or —CH(CH₃)₂;X is an iodine or bromine atom;R₅′ is a hydrogen or the group RCO, wherein R is hydrogen, branched orunbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy of 1 to 3 carbon atoms, or thioalkoxy of 1 to 5 carbon atoms; andQ is selected from the group consisting of —NNHCO—, —NNHCS—, —NNHCONH—,—NNHCSNH—, and —NO—;comprising the steps of:a. reacting a carboxylic acid of the formula

with a mercapto compound of the formula

H₂Q-Sp-SH

in an alcohol solvent in the presence of an alkyl carboxylic acid,alk²CO₂H where alk² is 1 to 4 carbon atoms at about 20° to 70° C. forabout 1 to 24 hours, wherein Alk¹, Sp¹, Ar, Z¹, Q, and Sp are as definedabove, to produce a bilinker-carboxylic acid of the formula,

b. isolating the bilinker-carboxylic acid of step (a);c. reacting the isolated bilinker-carboxylic acid of step (b) with an atleast of N-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol,pentafluorophenol, 4-nitrophenol, 2,4-dinitrophenol, orN-hydroxysulfosuccinimide in the presence of1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), orN,N′-disuccinimdyl carbonate in an inert solvent containing 0-50%N,N-dimethylformamide (DMF) to generate a trilinker-activated ester ofthe formula

where Z is hereinbefore defined;d. reacting the trilinker-activated ester in step c in the presence of abase or an organic base with CH₃—S—S—S—W′ in an inert organic solvent togenerate an activated ester of the formula

e. isolating the activated ester of step (d) and purifying to yieldcompounds of Formula (I)

The processes include a purifying of step comprising the use of areverse phase high performance liquid chromatography having a mobilephase of about pH 7.0 to 9.0 followed with a normal phasechromatography.

The processes include:

alk¹ is 3 carbon atoms;

Sp¹ is −O—;

Z¹ is methyl;Ar is unsubstituted 1,4-phenylene;Sp is 4 carbon atoms;

R₃ is H; Q is NNHC(O)—; R₂ is

R₄ is

R₅ is C₂H₅;

R₅′ is —C(O)—R;

R is methyl; and

Z is

The processes include but are not limited to Alk¹ is an alkylene of 2 to5 carbon atoms, and Sp¹ is an oxygen atom.

The processes include but are not limited Alk¹ is an alkylene of 3carbon atoms.

The processes include but are not limited Z¹ is alkyl of 1 to 3 carbonatoms.

The processes include but are not limited Ar is 1,2-, 1,3-, or1,4-phenylene, or 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-,or 2,7-naphthylidene.

The processes include but are not limited Ar is 1,4-phenylene.

The processes include but are not limited Q is —NNHCO—.

The processes include but are not limited Sp is straight orbranched-chain divalent or trivalent alkyl radical of 1 to 12 carbonatoms.

The processes include but are not limited Sp is straight orbranched-chain divalent or trivalent alkyl radical of 1 to 6 carbonatoms.

The processes include but are not limited the alcohol solvent ismethanol.

The processes include but are not limited the inert solvent isacetonitrile.

The processes include but are not limited alkyl carboxylic acid isacetic acid.

The processes include but are not limited the inert organic solvent isacetonitrile.

The processes include but are not limited Z is

Discussed herein is a process to prepare trilinker-activated esters ofthe formula:

wherein:Z is selected from the group consisting of

Alk¹ is a branched or unbranched alkylene chain of 2 to 6 carbon atoms;Sp¹ is selected from —S—, —O—, —CONH—, —NHCO—, and —NR′—;Z¹ is H, or alkyl of 1 to 5 carbon atoms;Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two,or three groups independently selected from alkyl of 1 to 6 carbonatoms, alkoxy of 1 to 5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms,halogen, nitro, —COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′,—O(CH₂)_(n)CONHR′, and —S(CH₂)_(n)CONHR′ or a 1,2-, 1,3-, 1,4-, 1,5-,1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionallysubstituted with one, two, three, or four groups independently selectedfrom alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 5 carbon atoms,thioalkoxy of 1 to 4 carbon atoms, halogen, nitro, —COOR′, —CONHR′,—O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′, —O(CH₂)_(n)CONHR′, and—S(CH₂)_(n)CONHR′;n is an integer from 0 to 5;R′ is a straight or branched alkyl of 1 to 5 carbon atoms optionallysubstituted by one or two groups of —OH, alkoxy of 1 to 4 carbon atoms,thioalkoxy of 1 to 4 carbon atoms;Sp is a straight or branched-chain divalent or trivalent alkyl radicalof 1 to 18 carbon atoms, divalent or trivalent aryl or heteroarylradical, divalent or trivalent cycloalkyl of 3 to 18 carbon atoms orheterocycloalkyl radical, divalent or trivalent aryl- orheteroaryl-alkyl (C₁-C₁₈) radical, divalent or trivalent cycloalkyl- orheterocyclo-alkyl-alkyl (C₁-C₁₈) radical or divalent or trivalentunsaturated alkyl radical of 2 to 18 carbon atoms, wherein heteroaryl isfuryl, thienyl, N-methylpyrrolyl, pyridinyl, N-methylimidazolyl,oxazolyl, pyrimidinyl, quinolyl, isoquinolyl, N-methylcarbazoyl,aminocoumarinyl, or phenazinyl and wherein if Sp is a trivalent radical,it can be additionally substituted by dialkylamino of 1 to 5 carbonatoms, alkoxy of 1 to 5 carbon atoms, hydroxy, or alkylthio of 1 to 5carbon atoms groups;

R₅ is —CH₃, —C₂H₅, or —CH(CH₃)₂;X is an iodine or bromine atom;R₅′ is a hydrogen or the group RCO, wherein R is hydrogen, branched orunbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy of 1 to 3 carbon atoms, or thioalkoxy of 1 to 5 carbon atoms; andQ is selected from the group consisting of —NNHCO—, —NNHCS—, —NHNCONH—,—NNCSNH—, and —NO—;comprising the steps of:a. reacting a carboxylic acid of the formula

with a mercapto compound of the formula

H₂Q-Sp-SH

in an alcohol solvent in the presence of an alkyl carboxylic acid,alk²CO₂H where alk² is 1 to 4 carbon atoms at about 20° to 70° C. forabout 1 to 24 hours, wherein Alk¹, Sp¹, Ar, Z¹, Q, and Sp are as definedabove, to produce a bilinker-carboxylic acid of the formula

b. isolating the bilinker-carboxylic acid of step (a);c. reacting the bilinker-carboxylic acid from step (b) in the presenceof a base or an organic base with CH₃—S—S—S—W′ in an inert organicsolvent to generate a bilinker-methyltrithio compound of the formula

d. reacting the isolated bilinker-methyltrithio compound of step (c)with N-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol, pentafluorophenol,4-nitrophenol, 2,4-dinitrophenol, or N-hydroxysulfosuccinimide in thepresence of 1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), orN,N′-disuccinimdyl carbonate in an inert solvent containing 0-50%N,N-dimethylformamide (DMF) to generate a trilinker-activated ester ofthe formula

Presented herein is a process for the preparation of trifunctionallinker intermediates, of the formula

wherein:Alk¹ is a branched or unbranched alkylene chain of 2 to 6 carbon atoms;Sp¹ is selected from —S—, —O—, —CONH—, —NHCO—, and —NR′—;Z¹ is H, or alkyl of 1 to 5 carbon atoms;Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two,or three groups independently selected from alkyl of 1 to 6 carbonatoms, alkoxy of 1 to 5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms,halogen, nitro, —COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′,—O(CH₂)_(n)CONHR′, and —S(CH₂)_(n)CONHR′ or a 1,2-, 1,3-, 1,4-, 1,5-,1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionallysubstituted with one, two, three, or four groups independently selectedfrom alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 5 carbon atoms,thioalkoxy of 1 to 4 carbon atoms, halogen, nitro, —COOR′, —CONHR′,—O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′, —O(CH₂)_(n)CONHR′, and—S(CH₂)_(n)CONHR′;n is an integer from 0 to 5;R′ is a straight or branched alkyl of 1 to 5 carbon atoms optionallysubstituted by one or two groups of —OH, alkoxy of 1 to 4 carbon atoms,thioalkoxy of 1 to 4 carbon atoms;Sp is a straight or branched-chain divalent or trivalent alkyl radicalof 1 to 18 carbon atoms, divalent or trivalent aryl or heteroarylradical, divalent or trivalent cycloalkyl of 3 to 18 carbon atoms orheterocycloalkyl radical, divalent or trivalent aryl- orheteroaryl-alkyl (C₁-C₁₈) radical, divalent or trivalent cycloalkyl- orheterocyclo-alkyl-alkyl (C₁-C₁₈) radical or divalent or trivalentunsaturated alkyl radical of 2 to 18 carbon atoms, wherein heteroaryl isfuryl, thienyl, N-methylpyrrolyl, pyridinyl, N-methylimidazolyl,oxazolyl, pyrimidinyl, quinolyl, isoquinolyl, N-methylcarbazoyl,aminocoumarinyl, or phenazinyl and wherein if Sp is a trivalent radical,it can be additionally substituted by dialkylamino of 1 to 5 carbonatoms, alkoxy of 1 to 5 carbon atoms, hydroxy, or alkylthio of 1 to 5carbon atoms groups;Q is selected from the group consisting of —NNHCO—, —NNHCS—, and—NNHCONH—;Z is selected from the group consisting of

comprising the steps of:a. reacting a carboxylic acid of the formula

with a mercapto compound of the formula

H₂Q-Sp-SH

in an alcohol solvent in the presence of an alkyl carboxylic acid,alk²CO₂H, where alk² is 1 to 4 carbon atoms at about 20° to 70° C. forabout 1 to 24 hours, wherein Alk¹, Sp¹, Ar, Z¹, Q, and Sp are as definedabove, to produce a bilinker-carboxylic acid of the formula

b. isolating the bilinker-carboxylic acid of step (a);c. reacting the isolated bilinker-carboxylic acid of step (b) withN-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol, pentafluorophenol,4-nitrophenol, 2,4-dinitrophenol, or N-hydroxysulfosuccinimide in thepresence of 1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), orN,N′-disuccinimdyl carbonate in an inert solvent containing 0-50% DMF togenerate trifunctional linker intermediates, of the formula

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compounds, compositions, andmethods of the invention and how to make and use them. Moreover, it willbe appreciated that the same thing can be said in more than one way.Consequently, alternative language and synonyms may be used for any oneor more of the terms discussed herein, nor is any special significanceto be placed upon whether or not a term is elaborated or discussedherein. The use of examples anywhere in this specification, includingexamples of any terms discussed herein, is illustrative only, and in noway limits the scope and meaning of the invention or of any exemplifiedterm. Likewise, the invention is not limited to the examples presented.

The terms, “about” or “approximately” shall generally mean within 20percent, preferably within 10 percent, and more preferably within 5percent of a given value or range.

The term “alkoxy” as used herein refers to an alkyl group, as definedabove, having an oxygen radical attached thereto. The term “thioalkoxy”refers to an alkoxy group as defined, having a sulfur radical attachedthereto.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In an embodiment, a straight chainor branched chain alkyl has 6 or fewer carbon atoms in its backbone. Theterm “alkyl” can be used alone or as part of a chemical name.

The term “aryl” is defined as an aromatic carbocyclic moiety and may besubstituted or unsubstituted. Aryl groups have 6 to 14 carbon atoms andinclude phenyl and napthyl. The term “aryl” also includes polycyclicring systems having two or more rings in which two or more carbons arecommon to two adjoining rings (the rings are “fused”) wherein at leastone of the carbocyclic rings is aromatic, e.g., the other rings can becycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls.

Carboxy is defined as a —CO₂H radical.

The term “halogen” or “halo” refers to an atom of fluorine, chlorine,bromine, or iodine.

The term “heteroaryl” refers to a 4 to 10 membered ring structure, whichring structure includes one to four heteroatoms. A heteroaryl comprisesa heterocyclic ring system of one to three fused rings, in which atleast one ring may have an aromatic character and contains 1 to 4heteroatoms the same or different selected from the group consisting ofS, N, and O. The remaining rings of the ring system may be fullyunsaturated, partially saturated, or fully saturated. Each ringcomprises three to ten members. The term “heteroatom” as used hereinmeans an atom of any element other than carbon or hydrogen and includefor example nitrogen, oxygen, sulfur, phosporus, and selenium.

The term “nitro” means —NO₂.

The term “substituted” is contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents of organic compounds include acyclic and cyclic, branchedand unbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

The term alkyl means a straight or branched alkyl of 1 to 18 carbonatoms, preferably 2 to 5 carbon atoms, 1 to 4 carbon atoms or 1 to 3carbon atoms. In some embodiments of the invention the term alkyl ismethyl.

The term alcohol solvent means methanol, ethanol and the like with aboiling point of less than about 100° C.

Alkylene refers to an alkyl, as defined above, which is a diradical,rather than a radical. An alkylene can be branched or unbranched and canhave 2-18 carbons.

Ambient temperature is about 25° C.

Inert solvent or inert organic solvent describes a solvent that willneither react or form a covalent bond in the steps to prepare compoundsof Formula (I), trilinker-activated esters or trifunctional linkerintermediates as described herein. An example of an inert solvent orinert organic solvent may be mixtures such as but not limited toacetonitrile/ethylacetate (1:1)), dichloromethane,N,N-dimethylformamide, tetrahydrofuran, dioxane or acetonitrile. Aninert solvent may contain 0-50% N,N-dimethylformamide. An inert organicsolvent is for example acetonitrile, ethyl acetate or dichloromethane.

An organic base is for example an alkylamine base which includestriethylamine, N,N-diethylmethylamine, N,N-diethylaniline,N,N-diethylethylenediamine, N,N-diisopropylethylamine, tributylamine ortripropylamine and further include dimethylaminopyridine(DMAP) withdiisopropylethylamine (DIEA), N-methylmorpholine, N-methylpyrrolidine,2,6-di-tertbutyl-4-methylpyridine or pyridine. A base is an alkali metalhydroxide, alkali metal carbonate or alkali metal bicarbonate.

DETAILED DESCRIPTION OF THE INVENTION

The process for the preparation of calicheamicin derivatives of FormulaI and tri and bifunctional linker intermediates useful in thepreparation of said derivatives of the present invention are describedin the following reaction Schemes I and II.

In accordance with Scheme I, a carboxylic acid 1 wherein Alk¹, Sp¹, Arand Z¹ are hereinbefore defined, found in U.S. Pat. No. 5,773,001, whichis hereby incorporated herein by reference, are condensed with mercaptocompound 2 where Sp and Q are hereinbefore defined in an alcoholicsolvent with a boiling point of less than about 100° C. in the presenceof an alkyl carboxylic acid in a about 5% acetic acid at about 20° toabout 70° C. for about 1 to about 24 hours, to affordbilinker-carboxylic acid 3 wherein Alk¹, Sp¹, Ar, Q, Sp and Z¹ are asdefined above.

Bilinker-carboxylic acid 3 is reacted with N-hydroxysuccinimide,2,3,5,6-tetrafluorophenol, pentafluorophenol, 4-nitrophenol,2,4-dinitrophenol, or N-hydroxysulfosuccinimide in the presence of1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), or othercarbodiimide or N,N′-disuccinimdyl carbonate in an inert solvent such asdichloromethane, tetrahydrofuran, dioxane, or acetonitrile containing0-50% DMF or DMF to generate trilinker-activated ester 4 where Z isselected from the group consisting of

For example, reaction of bilinker-carboxylic acid 3 with a couplingagent, such as 1,3-dicyclohexylcarbodiimide(DCC) or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, andN-hydroxysuccinimide or other comparable carboxyl-activating group in aninert solvent, such as N,N-dimethylformamide (DMF), tetrahydrofuran,dioxane or acetonitrile, leads to the formation of a trilinker-activatedester 4, such as the N-hydroxysuccinimide ester described herein.Preferred is N-hydroxysuccinimide, DCC at ambient temperature indioxane. A preferred solvent mixture is acetonitrile containing 0-50%DMF. Reaction of the bilinker-carboxylic acid 3 withN-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol, pentafluorophenol,4-nitrophenol, 2,4-dinitrophenol, or N-hydroxysulfosuccinimide in thepresence of 1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), or othercarbodiimide in an inert solvent such as dioxane or acetonitrilecontaining 0-50% N,N-dimethylformamide (DMF) leads to the formation of atrilinker-activated ester 4. The trilinker-activated ester 4 can beisolated by removal of the volatile solvents and further purified byreverse or normal phase chromatography on an inert support whichincludes silica-60.

Trilinker-activated ester 4 is first reacted with an alkali metalcarbonate which includes but is not limited to sodium carbonate andforms the sodium salt of trilinker-activated ester 4 in acetonitrile byheating at gentle reflux. Further reaction of the sodium salt oftrilinker-activated ester 4 with methyltrithioantitumor antibiotic 5 atabout −15° C. in an inert organic solvent, preferably acetonitrile givesactivated ester 6 wherein Z, Alk¹, Sp¹, Ar, Z¹, Q, Sp and W′ arehereinbefore defined. In particular, N-acetyl-LL-E33288 γ₁ ^(I) is thepreferred methyltrithioantitumor antibiotic 5. Preferred is the reactionin acetonitrile at about 0° C. Optionally an organic base may replacethe alkali metal carbonate which preferably includes triethylamine, inacetonitrile at about 0° C.

As further described in Scheme II, reaction of bilinker-carboxylic 3prepared by condensation of carboxylic acid 1 wherein Alk¹, Sp¹, Ar andZ¹ are hereinbefore defined, with mercapto compound 2 according toscheme I, is reacted with methyltrithioantitumor antibiotic 5 in thepresence of triethylamine in N,N-dimethylformamide (DMF) at about −5° C.affords intermediate 7 which is further converted to trilinker-activatedester 6 by reaction with N-hydroxysuccinimide,2,3,5,6-tetrafluorophenol, pentafluorophenol, 4-nitrophenol,2,4-dinitrophenol, or N-hydroxysulfosuccinimide in the presence of1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), or othercarbodiimide or N,N′-disuccinimdyl carbonate in an inert solvent mixtureof DMF and acetonitrile which is then purified preferably bychromatography to afford antitumor antibiotics of Formula (I).

The following examples are presented to illustrate certain embodimentsof the present invention, but should not be construed as limiting thescope of this invention. Those skilled in the art will readilyunderstand that known variations of the conditions of the followingpreparative procedures can be used to prepare these compounds.

Example 1 Butanoic Acid,3-mercapto-3-methyl-,2[(E)-1-[4-(4-hydroxy-4-oxobutoxy)phenyl]ethylidene]hydrazide

To a stirred mixture of 0.5 g [3.4 mmol] of 3-methyl-3-mercapto-butanoicacid hydrazide in 5.0 ml of methanol is rapidly added 0.91 g [4.1 mmol]of 4-(4-acetylphenoxy)-butanoic acid followed by an additional 10 ml ofmethanol and 1.5 ml of acetic acid and stirring continued for 24 hours.The reaction mixture is filtered and the solid washed with 100 ml ofmethanol to give 0.78 g of the title compound as a solid.

Example 2 Butanoic Acid,3-mercapto-3-methyl-,2-[(E)-1-[4-(4-hydroxy-4-oxobutoxy)phenyl]ethylidene]hydrazide

A mixture of 3-methyl-3-mercapto-butanoic acid hydrazide (4.0 g, 27mmol), 4-(4-acetylphenoxy)-butanoic acid (5.0 g, 22.5 mmol) and aceticacid (7.5 mL) in methyl alcohol (75 mL) is heated at about 45° C. forabout 7 h. The mixture is allowed to cool to room temperature. The whitesolid (7.12 g, 90%) is collected on a Buchner funnel and washed withMeOH (2×10 mL). ¹H NMR (DMSO-d₆): δ (ppm) 12.14 (s, 1H), 10.37 and 10.21(s, 1H), 7.74-7.70 (m, 2H), 6.97-6.95 (m, 2H), 4.04 (t, 2H), 3.09 and3.04 (s, 2H), 2.66 (s, 1H), 2.41-2.37 (t, 2H), 2.22 and 2.20 (s, 3H),1.97-1.93 (m, 2H), 1.8 (s, 3H), 1.47 (s, 3H). MS: 375 (M⁺+Na).

Example 3 Butanoic Acid,3-Mercapto-3-Methyl-,2-[(E)-1-[4-[4-[(2,5-Dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide

To a mixture of 0.5 g [1.42 mmol] of butanoic acid,3-mercapto-3-methyl-,2-[(E)-1-[4-(4-hydroxy-4-oxobutoxy)phenyl]ethylidene]hydrazide (Example 1 or 2) and 0.22 g [1.89 mmol] ofN-hydroxysuccinimide is added 10 ml of N,N-dimethylformamide followed bythe rapid addition of 0.70 g [3.65 mmol] of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and themixture stirred at room temperature for 3 hours. The reaction mixture isconcentrated in vacuo to a residue which is partitioned between ethylacetate and water. The separated organic layer is washed with water,saturated sodium chloride and dried (MgSO₄). The organic layer isevaporated in vacuo to give an oily residue which crystallized fromethyl acetate-hexane affording 0.21 g of the title compound as acolorless solid.

Example 4 Butanoic Acid,3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide

A mixture of butanoic acid,3-mercapto-3-methyl-,2-[(E)-1-[4-(4-hydroxy-4-oxobutoxy)phenyl]ethylidene]hydrazide(Examples 1 or 2) (3.69 g, 10.48 mmol) and N-hydroxysuccinimide (1.386g, 12.05 mmol) is suspended in dioxane (60 mL), DCC (2.482 g, 12.05mmol) in dioxane (30 mL) is added dropwise over 15 min. The mixture isstirred at room temperature for 24 h. The precipitated dicyclohexylureais filtered off and washed with dioxane (2×10 mL). The filtrate isconcentrated to about 50 mL and while stirring, water (250 mL) is added.The resulting white solid is collected on a Buchner funnel, washed withwater (2×50 mL), and dried in vacuo at room temperature. To this whitesolid is added MeCN (60 mL) and the mixture is heated at 50° C. until itbecame solution, isopropyl alcohol (IPA) (400 mL) is added. The mixtureis then cooled to 0-5° C. for 2 h. The solid is collected on a Buchnerfunnel, washed with cold IPA (2×20 mL) an dried in vacuo to afford theproduct of the example as a white solid (3.58 g, 70%). MS: 450 (M⁺+1).

Example 5 Butanoic Acid,3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazidecondensed with N-acetyl-LL-E33288γ₁ ^(I)

To a stirred solution of 0.505 g [1.12 mmol] butanoic acid,3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide(Example 4) in 50 ml of acetonitrile is added 0.123 g [1.16 mmol] ofsodium carbonate followed by heating at gentle reflux for 1 hour, cooledto room temperature and filtered. The filtrate is cooled to −15° C. anda solution of 1.4969 g [1.1 mmol] of N-acetyl-LL-E33288 γ₁ ^(I) in 5 mlof acetonitrile added slowly by dropwise addition over 20 minutes andstirring continued for about 1.5 hours. The reaction mixture is allowedto warm to room temperature and stirred for about 3 hours. The volatilesare evaporated in vacuo to a residue which is stored in a freezer. Tothe residue is added 25 ml of ethyl acetate followed by storage in afreezer for about 1 hour. The reaction mixture is filtered and the ethylacetate evaporated to a residue which is dissolved in 10 ml of ethylacetate and applied to a column of 110 g of silica gel. The column iseluted with 1-5% methyl alcohol in ethyl acetate to give 0.322 g of thedesired product having 65.27% purity as determined by HPLC.

Example 6 Butanoic Acid,3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazideCondensed with N-acetyl-LL-E33288γ₁ ^(I)

A solution of butanoic acid,3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide(450 mg, 1 mmol) (Example 4) in CH₃CN (100 mL) containing Et₃N (0.35 mL)is treated with a solution of N-acetyl-LL-E33288 γ₁ ^(I) (500 mg, 0.355mmol) in CH₃CN (100 mL) at 0-5° C. The mixture is then stirred foranother 1 h while cooling with a ice-bath. The solvent is removed andthe residue is purified on a silica gel column eluting with CH₂Cl₂-MeOHto afford the product of the example (340 mg, 54%) as a white solid. MS:1780 (M⁺+1)

Example 7 Butanoic Acid,3-mercapto-3-methyl-,2[(E)-1-[4-(4-hydroxy-4-oxobutoxy)phenyl]ethylidene]hydrazideCondensed with N-acetyl-LL-E33288γ₁ ^(I)

To a stirring solution of N-acetyl-LL-E33288 γ₁ ^(I) (200 mg, 0.142mmol) in 10 ml acetonitrile/ethyl acetate (1:1) at −5° C. is added in 1ml aliquots every 10 min a solution of butanoic acid,3-mercapto-3-methyl-,2-[(E)-1-[4-(4-hydroxy-4-oxobutoxy)phenyl]ethylidene]hydrazide(150 mg, 0.43 mmol) (Examples 1 or 2) in 10 ml acetonitrile/ethylacetate (1:1) and 0.06 ml triethyl amine. The solution is stirred fortwo hours at −5° C. after the last addition of the butanoic acid,3-mercapto-3-methyl-,2-[(E)-1-[4-(4-hydroxy-4-oxobutoxy)phenyl]ethylidene]hydrazidesolution. The solvent was removed under reduced pressure and the residueis purified on a silica gel column eluting with CH₂Cl₂-MeOH to affordthe product of the example as a white solid. MS 1684 (M⁺+1)

Example 8 Butanoic Acid,3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazidecondensed with N-acetyl-LL-E33288γ₁ ^(I)

To a stirring solution of butanoic acid,3-mercapto-3-methyl-,2[(E)-1-[4-(4-hydroxy-4-oxobutoxy)phenyl]ethylidene]hydrazidewith N-acetyl-LL-E33288γ₁ ^(I) (100 mg, 0.059 mmol) in 0.5 mL of DMF and1.8 mL of acetonitrile at 25° C. is added N-hydroxysuccinimide (236 mg,2.05 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (160 mg0.835 mmol). Following the addition, the solution is stirred for onehour at 25° C. The acetonitrile is removed under reduced pressure andthe resulting DMF solution is added to 3 mL of stirring water giving aprecipitate. The precipitate is filtered, dried and purified on a silicagel column eluting with CH₂Cl₂-isopropyl alcohol giving the product ofthe example (53 mg, 50%) obtained as a white solid. MS: 1780 (M⁺+1)

What is claimed is:
 1. A process to prepare compounds of Formula (I):

wherein: Z is selected from the group consisting of

Alk¹ is a branched or unbranched alkylene chain of 2 to 6 carbon atoms;Sp¹ is selected from —S—, —O—, —CONH—, —NHCO—, and —NR′—; Z¹ is H, oralkyl of 1 to 5 carbon atoms; Ar is 1,2-, 1,3-, or 1,4-phenyleneoptionally substituted with one, two, or three groups independentlyselected from alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 5 carbonatoms, thioalkoxy of 1 to 4 carbon atoms, halogen, nitro, —COOR′,—CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′, —O(CH₂)_(n)CONHR′, and—S(CH₂)_(n)CONHR′ or a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-,2,6-, or 2,7-naphthylidene optionally substituted with one, two, three,or four groups independently selected from alkyl of 1 to 6 carbon atoms,alkoxy of 1 to 5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms,halogen, nitro, —COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′,—O(CH₂)_(n)CONHR′, and —S(CH₂)_(n)CONHR′; n is an integer from 0 to 5;R′ is a straight or branched alkyl of 1 to 5 carbon atoms optionallysubstituted by one or two groups of —OH, alkoxy of 1 to 4 carbon atoms,thioalkoxy of 1 to 4 carbon atoms; Sp is a straight or branched-chaindivalent or trivalent alkyl radical of 1 to 18 carbon atoms, divalent ortrivalent aryl or heteroaryl radical, divalent or trivalent cycloalkylof 3 to 18 carbon atoms or heterocycloalkyl radical, divalent ortrivalent aryl- or heteroaryl-alkyl (C₁-C₁₈) radical, divalent ortrivalent cycloalkyl- or heterocyclo-alkyl-alkyl (C₁-C₁₈) radical ordivalent or trivalent unsaturated alkyl radical of 2 to 18 carbon atoms,wherein heteroaryl is furyl, thienyl, N-methylpyrrolyl, pyridinyl,N-methylimidazolyl, oxazolyl, pyrimidinyl, quinolyl, isoquinolyl,N-methylcarbazoyl, aminocoumarinyl, or phenazinyl and wherein if Sp is atrivalent radical, it can be additionally substituted by dialkylamino of1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms, hydroxy, oralkylthio of 1 to 5 carbon atoms groups;

R₅ is —CH₃, —C₂H₅, or —CH(CH₃)₂; X is an iodine or bromine atom; R₅′ isa hydrogen or the group RCO, wherein R is hydrogen, branched orunbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy of 1 to 3 carbon atoms, or thioalkoxy of 1 to 5 carbon atoms; andQ is selected from the group consisting of —NNHCO—, —NNHCS—, —NNHCONH—,—NNHCSNH—, and —NO—; comprising the steps of: a. reacting a carboxylicacid of the formula

with a mercapto compound of the formulaH₂Q-Sp-SH in an alcohol solvent in the presence of an alkyl carboxylicacid, alk²CO₂H where alk² is 1 to 4 carbon atoms at about 20° to 70° C.for about 1 to 24 hours, wherein Alk¹, Sp¹, Ar, Z¹, Q, and Sp are asdefined above, to produce a bilinker-carboxylic acid of the formula,wherein the mercapto compound and carboxylic acid are present in a ratioof about 1.2:1

b. isolating the bilinker-carboxylic acid of step (a); c. reacting theisolated bilinker-carboxylic acid of step (b) with an at least a 3 foldmolar excess of N-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol,pentafluorophenol, 4-nitrophenol, 2,4-dinitrophenol, orN-hydroxysulfosuccinimide in the presence of1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), orN,N′-disuccinimdyl carbonate in an inert solvent containing 0-50%N,N-dimethylformamide (DMF) to generate a trilinker-activated ester ofthe formula

where Z is hereinbefore defined; d. reacting the trilinker-activatedester formed in step c with a methyltrithio antitumor antiobioticCH₃—S—S—S—W′ in the presence of a base or an organic base with amethyltrithio antitumor antibiotic CH₃—S—S—S—W′ in an inert organicsolvent to generate an activated ester of the formula below, wherein thetrilinker-activated ester in step c and the CH₃—S—S—S—W′ are in a ratioof 3.3:1 and the temperature of the reaction is ≦5° C.

e. isolating the activated ester of step (d) and purifying to yieldantitumor antibiotics of Formula (I)


2. The process of claim 1 wherein the purifying of step (e) comprisesthe use of reverse phase high performance liquid chromatography having amobile phase of about pH 7.0 to 9.0 followed by normal phasechromatography.
 3. A process according to claim 1, wherein alk¹ is 3carbon atoms; Sp¹ is —O—; Z¹ is methyl; Ar is unsubstituted1,4-phenylene; Sp is 4 carbon atoms; R₃ is H; Q is NNHC(O)—; R₂ is

R₄ is

R₅ is C₂H₅; R₅′ is —C(O)—R; R is methyl; and Z is


4. A process to prepare antitumor antibiotics of Formula (I):

wherein: Z is selected from the group consisting of

Alk¹ is a branched or unbranched alkylene chain of 2 to 6 carbon atoms;Sp¹ is selected from —S—, —O—, —CONH—, —NHCO—, and —NR′—; Z¹ is H, oralkyl of 1 to 5 carbon atoms; Ar is 1,2-, 1,3-, or 1,4-phenyleneoptionally substituted with one, two, or three groups independentlyselected from alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 5 carbonatoms, thioalkoxy of 1 to 4 carbon atoms, halogen, nitro, —COOR′,—CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′, —O(CH₂)_(n)CONHR′, and—S(CH₂)_(n)CONHR′ ora 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-,2,6-, or 2,7-naphthylidene optionally substituted with one, two, three,or four groups independently selected from alkyl of 1 to 6 carbon atoms,alkoxy of 1 to 5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms,halogen, nitro, —COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′,—O(CH₂)_(n)CONHR′, and —S(CH₂)_(n)CONHR′; n is an integer from 0 to 5;R′ is a straight or branched alkyl of 1 to 5 carbon atoms optionallysubstituted by one or two groups of —OH, alkoxy of 1 to 4 carbon atoms,thioalkoxy of 1 to 4 carbon atoms; Sp is a straight or branched-chaindivalent or trivalent alkyl radical of 1 to 18 carbon atoms, divalent ortrivalent aryl or heteroaryl radical, divalent or trivalent cycloalkylof 3 to 18 carbon atoms or heterocycloalkyl radical, divalent ortrivalent aryl- or heteroaryl-alkyl (C₁-C₁₈) radical, divalent ortrivalent cycloalkyl- or heterocyclo-alkyl-alkyl (C₁-C₁₈) radical ordivalent or trivalent unsaturated alkyl radical of 2 to 18 carbon atoms,wherein heteroaryl is furyl, thienyl, N-methylpyrrolyl, pyridinyl,N-methylimidazolyl, oxazolyl, pyrimidinyl, quinolyl, isoquinolyl,N-methylcarbazoyl, aminocoumarinyl, or phenazinyl and wherein if Sp is atrivalent radical, it can be additionally substituted by dialkylamino of1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms, hydroxy, oralkylthio of 1 to 5 carbon atoms groups;

R₅ is —CH₃, —C₂H₅, or —CH(CH₃)₂; X is an iodine or bromine atom; R₅′ isa hydrogen or the group RCO, wherein R is hydrogen, branched orunbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy of 1 to 3 carbon atoms, or thioalkoxy of 1 to 5 carbon atoms; andQ is selected from the group consisting of —NNHCO—, —NNHCS—, —NNHCONH—,—NNHCSNH—, and —NO—; comprising the steps of: a. reacting a carboxylicacid of the formula

with a mercapto compound of the formulaH₂Q-Sp-SH in an alcohol solvent in the presence of an alkyl carboxylicacid, alk²CO₂H where alk² is 1 to 4 carbon atoms at about 20° to 70° C.for about 1 to 24 hours, wherein Alk¹, Sp¹, Ar, Z¹, Q, and Sp are asdefined above, to produce a bilinker-carboxylic acid of the formula,wherein the mercapto compound and carboxylic acid are present in a ratioof about 1.2:1

b. isolating the bilinker-carboxylic acid of step (a); c. reacting theisolated bilinker-carboxylic acid of step (b) with a methyltrithioantitumor antibiotic CH₃—S—S—S—W′ in the presence of a base or anorganic base with a methyltrithio antitumor antibiotic CH₃—S—S—S—W′ inan inert organic solvent; d. reacting the compound of step (c) with anat least a 3 fold molar excess of N-hydroxysuccinimide,2,3,5,6-tetrafluorophenol, pentafluorophenol, 4-nitrophenol,2,4-dinitrophenol, or N-hydroxysulfosuccinimide in the presence of1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), orN,N′-disuccinimdyl carbonate in an inert solvent containing 0-50%N,N-dimethylformamide (DMF) and purifying to yield antitumor antibioticsof Formula (I)


5. The process of claim 4 wherein the purifying of step (e) comprisesthe use of a reverse phase high performance liquid chromatography havinga mobile phase of about pH 7.0 to 9.0 followed with a normal phasechromatography.
 6. A process according to claim 4, wherein alk¹ is 3carbon atoms; Sp¹ is —O—; Z¹ is methyl; Ar is unsubstituted1,4-phenylene; Sp is 4 carbon atoms; R₃ is H; Q is NNHC(O)—; R₂ is

R₄ is

R₅ is C₂H₅; R₅′ is —C(O)—R; R is methyl; and Z is


7. A process to prepare antitumor antibiotics of Formula (I):

wherein: Z is selected from the group consisting of

Alk¹ is a branched or unbranched alkylene chain of 2 to 6 carbon atoms;Sp¹ is selected from —S—, —O—, —CONH—, —NHCO—, and —NR′—; Z¹ is H, oralkyl of 1 to 5 carbon atoms; Ar is 1,2-, 1,3-, or 1,4-phenyleneoptionally substituted with one, two, or three groups independentlyselected from alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 5 carbonatoms, thioalkoxy of 1 to 4 carbon atoms, halogen, nitro, —COOR′,—CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′, —O(CH₂)_(n)CONHR′, and—S(CH₂)_(n)CONHR′ or a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-,2,6-, or 2,7-naphthylidene optionally substituted with one, two, three,or four groups independently selected from alkyl of 1 to 6 carbon atoms,alkoxy of 1 to 5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms,halogen, nitro, —COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′,—O(CH₂)_(n)CONHR′, and —S(CH₂)_(n)CONHR′; n is an integer from 0 to 5;R′ is a straight or branched alkyl of 1 to 5 carbon atoms optionallysubstituted by one or two groups of —OH, alkoxy of 1 to 4 carbon atoms,thioalkoxy of 1 to 4 carbon atoms; Sp is a straight or branched-chaindivalent or trivalent alkyl radical of 1 to 18 carbon atoms, divalent ortrivalent aryl or heteroaryl radical, divalent or trivalent cycloalkylof 3 to 18 carbon atoms or heterocycloalkyl radical, divalent ortrivalent aryl- or heteroaryl-alkyl (C₁-C₁₈) radical, divalent ortrivalent cycloalkyl- or heterocyclo-alkyl-alkyl (C₁-C₁₈) radical ordivalent or trivalent unsaturated alkyl radical of 2 to 18 carbon atoms,wherein heteroaryl is furyl, thienyl, N-methylpyrrolyl, pyridinyl,N-methylimidazolyl, oxazolyl, pyrimidinyl, quinolyl, isoquinolyl,N-methylcarbazoyl, aminocoumarinyl, or phenazinyl and wherein if Sp is atrivalent radical, it can be additionally substituted by dialkylamino of1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms, hydroxy, oralkylthio of 1 to 5 carbon atoms groups;

R₅ is —CH₃, —C₂H₅, or —CH(CH₃)₂; X is an iodine or bromine atom; R₅′ isa hydrogen or the group RCO, wherein R is hydrogen, branched orunbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy of 1 to 3 carbon atoms, or thioalkoxy of 1 to 5 carbon atoms; andQ is selected from the group consisting of —NNHCO—, —NNHCS—, —NNHCONH—,—NNHCSNH—, and —NO—; comprising the steps of: a. reacting a carboxylicacid of the formula

with a mercapto compound of the formulaH₂Q-Sp-SH in an alcohol solvent in the presence of an alkyl carboxylicacid, alk²CO₂H where alk² is 1 to 4 carbon atoms at about 20° to 70° C.for about 1 to 24 hours, wherein Alk¹, Sp¹, Ar, Z¹, Q, and Sp are asdefined above, to produce a bilinker-carboxylic acid of the formula,

b. isolating the bilinker-carboxylic acid of step (a); c. reacting theisolated bilinker-carboxylic acid of step (b) with an at least ofN-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol, pentafluorophenol,4-nitrophenol, 2,4-dinitrophenol, or N-hydroxysulfosuccinimide in thepresence of 1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), orN,N′-disuccinimdyl carbonate in an inert solvent containing 0-50%N,N-dimethylformamide (DMF) to generate a trilinker-activated ester ofthe formula

where Z is hereinbefore defined; d. reacting the trilinker-activatedester in step c in the presence of a base or an organic base with amethyltrithio antitumor antibiotic CH₃—S—S—S—W′ in an inert organicsolvent to generate an activated ester of the formula

e. isolating the activated ester of step (d) and purifying to yieldantitumor antibiotics of Formula (I)


8. A process according to claim 1, wherein alk¹ is 3 carbon atoms; Sp¹is —O—; Z¹ is methyl; Ar is unsubstituted 1,4-phenylene; Sp is 4 carbonatoms; R₃ is H; Q is NNHC(O)—; R₂ is

R₄ is

R₅ is C₂H₅; R₅′ is —C(O)—R; R is methyl; and Z is


9. A process according to claim 1, wherein Alk¹ is an alkylene of 2 to 5carbon atoms, and Sp¹ is an oxygen atom.
 10. A process according toclaim 9, wherein Alk¹ is an alkylene of 3 carbon atoms.
 11. A processaccording to claim 1, wherein Z¹ is alkyl of 1 to 3 carbon atoms.
 12. Aprocess according to claim 1, wherein Ar is 1,2-, 1,3-, or1,4-phenylene, or 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-,or 2,7-naphthylidene.
 13. A process according to claim 12, wherein Ar is1,4-phenylene.
 14. A process according to claim 1, wherein Q is —NNHCO—.15. A process according to claim 1, wherein Sp is straight orbranched-chain divalent or trivalent alkyl radical of 1 to 12 carbonatoms.
 16. A process according to claim 15, wherein Sp is straight orbranched-chain divalent or trivalent alkyl radical of 1 to 6 carbonatoms.
 17. The process according to claim 1, wherein the alcohol solventis methanol.
 18. The process according to claim 1, wherein the inertsolvent is acetonitrile.
 19. The process according to claim 1, whereinalkyl carboxylic acid is acetic acid.
 20. The process according to claim1, wherein the inert organic solvent is acetonitrile.
 21. A processaccording to claim 1, wherein Z is


22. A process to prepare trilinker-activated esters of the formula:

wherein: Z is selected from the group consisting of

Alk¹ is a branched or unbranched alkylene chain of 2 to 6 carbon atoms;Sp¹ is selected from —S—, —O—, —CONH—, —NHCO—, and —NR′—; Z¹ is H, oralkyl of 1 to 5 carbon atoms; Ar is 1,2-, 1,3-, or 1,4-phenyleneoptionally substituted with one, two, or three groups independentlyselected from alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 5 carbonatoms, thioalkoxy of 1 to 4 carbon atoms, halogen, nitro, —COOR′,—CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′, —O(CH₂)_(n)CONHR′, and—S(CH₂)_(n)CONHR′ or a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-,2,6-, or 2,7-naphthylidene optionally substituted with one, two, three,or four groups independently selected from alkyl of 1 to 6 carbon atoms,alkoxy of 1 to 5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms,halogen, nitro, —COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′,—O(CH₂)_(n)CONHR′, and —S(CH₂)_(n)CONHR′; n is an integer from 0 to 5;R′ is a straight or branched alkyl of 1 to 5 carbon atoms optionallysubstituted by one or two groups of —OH, alkoxy of 1 to 4 carbon atoms,thioalkoxy of 1 to 4 carbon atoms; Sp is a straight or branched-chaindivalent or trivalent alkyl radical of 1 to 18 carbon atoms, divalent ortrivalent aryl or heteroaryl radical, divalent or trivalent cycloalkylof 3 to 18 carbon atoms or heterocycloalkyl radical, divalent ortrivalent aryl- or heteroaryl-alkyl (C₁-C₁₈) radical, divalent ortrivalent cycloalkyl- or heterocyclo-alkyl-alkyl (C₁-C₁₈) radical ordivalent or trivalent unsaturated alkyl radical of 2 to 18 carbon atoms,wherein heteroaryl is furyl, thienyl, N-methylpyrrolyl, pyridinyl,N-methylimidazolyl, oxazolyl, pyrimidinyl, quinolyl, isoquinolyl,N-methylcarbazoyl, aminocoumarinyl, or phenazinyl and wherein if Sp is atrivalent radical, it can be additionally substituted by dialkylamino of1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms, hydroxy, oralkylthio of 1 to 5 carbon atoms groups;

R₅ is —CH₃, —C₂H₅, or —CH(CH₃)₂; X is an iodine or bromine atom; R₅′ isa hydrogen or the group RCO, wherein R is hydrogen, branched orunbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy of 1 to 3 carbon atoms, or thioalkoxy of 1 to 5 carbon atoms; andQ is selected from the group consisting of —NNHCO—, —NNHCS—, —NHNCONH—,—NNCSNH—, and —NO—; comprising the steps of: a. reacting a carboxylicacid of the formula

with a mercapto compound of the formulaH₂Q-Sp-SH in an alcohol solvent in the presence of an alkyl carboxylicacid, alk²CO₂H where alk² is 1 to 4 carbon atoms at about 20° to 70° C.for about 1 to 24 hours, wherein Alk¹, Sp¹, Ar, Z¹, Q, and Sp are asdefined above, to produce a bilinker-carboxylic acid of the formula

b. isolating the bilinker-carboxylic acid of step (a); c. reacting thebilinker-carboxylic acid from step (b) in the presence of a base or anorganic base with a methyltrithio antitumor antibiotic CH₃—S—S—S—W′ inan inert organic solvent to generate a bilinker-methyltrithio antitumorantibiotic of the formula

d. reacting the isolated bilinker-methyltrithio antitumor antibiotic ofstep (c) with N-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol,pentafluorophenol, 4-nitrophenol, 2,4-dinitrophenol, orN-hydroxysulfosuccinimide in the presence of1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), orN,N′-disuccinimdyl carbonate in an inert solvent containing 0-50%N,N-dimethylformamide (DMF) to generate a trilinker-activated ester ofthe formula


23. The process according to claim 17, wherein Alk¹ is an alkylene of 2to 5 carbon atoms, and Sp¹ is an oxygen atom.
 24. A process for thepreparation of trifunctional linker intermediates, of the formula

wherein: Alk¹ is a branched or unbranched alkylene chain of 2 to 6carbon atoms; Sp¹ is selected from —S—, —O—, —CONH—, —NHCO—, and —NR′—;Z¹ is H, or alkyl of 1 to 5 carbon atoms; Ar is 1,2-, 1,3-, or1,4-phenylene optionally substituted with one, two, or three groupsindependently selected from alkyl of 1 to 6 carbon atoms, alkoxy of 1 to5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms, halogen, nitro,—COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′, —O(CH₂)_(n)CONHR′,and —S(CH₂)_(n)CONHR′ or a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-,2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one, two,three, or four groups independently selected from alkyl of 1 to 6 carbonatoms, alkoxy of 1 to 5 carbon atoms, thioalkoxy of 1 to 4 carbon atoms,halogen, nitro, —COOR′, —CONHR′, —O(CH₂)_(n)COOR′, —S(CH₂)_(n)COOR′,—O(CH₂)_(n)CONHR′, and —S(CH₂)_(n)CONHR′; n is an integer from 0 to 5;R′ is a straight or branched alkyl of 1 to 5 carbon atoms optionallysubstituted by one or two groups of —OH, alkoxy of 1 to 4 carbon atoms,thioalkoxy of 1 to 4 carbon atoms; Sp is a straight or branched-chaindivalent or trivalent alkyl radical of 1 to 18 carbon atoms, divalent ortrivalent aryl or heteroaryl radical, divalent or trivalent cycloalkylof 3 to 18 carbon atoms or heterocycloalkyl radical, divalent ortrivalent aryl- or heteroaryl-alkyl (C₁-C₁₈) radical, divalent ortrivalent cycloalkyl- or heterocyclo-alkyl-alkyl (C₁-C₁₈) radical ordivalent or trivalent unsaturated alkyl radical of 2 to 18 carbon atoms,wherein heteroaryl is furyl, thienyl, N-methylpyrrolyl, pyridinyl,N-methylimidazolyl, oxazolyl, pyrimidinyl, quinolyl, isoquinolyl,N-methylcarbazoyl, aminocoumarinyl, or phenazinyl and wherein if Sp is atrivalent radical, it can be additionally substituted by dialkylamino of1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms, hydroxy, oralkylthio of 1 to 5 carbon atoms groups; Q is selected from the groupconsisting of —NNHCO—, —NNHCS—, and —NNHCONH—; Z is selected from thegroup consisting of

comprising the steps of: a. reacting a carboxylic acid of the formula

with a mercapto compound of the formulaH₂Q-Sp-SH in an alcohol solvent in the presence of an alkyl carboxylicacid, alk²CO₂H, where alk² is 1 to 4 carbon atoms at about 20° to 70° C.for about 1 to 24 hours, wherein Alk¹, Sp¹, Ar, Z¹, Q, and Sp are asdefined above, to produce a bilinker-carboxylic acid of the formula

b. isolating the bilinker-carboxylic acid of step (a); c. reacting theisolated bilinker-carboxylic acid of step (b) with N-hydroxysuccinimide,2,3,5,6-tetrafluorophenol, pentafluorophenol, 4-nitrophenol,2,4-dinitrophenol, or N-hydroxysulfosuccinimide in the presence of1,3-dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), orN,N′-disuccinimdyl carbonate in an inert solvent containing 0-50% DMF togenerate trifunctional linker intermediates, of the formula